Genetic and biochemical analysis of Col and R plasmids with particular emphasis on the plasmids Co1E1, R6K and R100 and in vitro generated derivatives of Co1E1 carrying unrelated specific regions of prokaryotic DNA. Temperature-sensitive mutants defective in Co1E1 replication will be analyzed at a non-permissive temperature for defects in relaxation complex, formation of RNA-containing DNA and the generation of replicative intermediates. Additional evidence in support of the proposed covalent DNA-protein linkage will be sought. Attempts will be made to determine the origin and directionality of replication of various plasmids and the role of RNA priming in the synthesis of these elements. An electron microscopy analysis of plasmid DNA-membrane complexes will be carried out to probe the role of membranes in plasmid DNA replication. The role of relaxation complex in the replication and conjugal transfer of plasmids also will be emphasized. Finally, reconstitution of plasmids will be carried out in vitro to assess the value of Co1E1 interaction of replicons in a composite molecule.